E8 Promoter Research Articles

A pear S1-bZIP transcription factor PpbZIP44 modulates carbohydrate metabolism, amino acid, and flavonoid accumulation in fruits.

Fruit quality is defined by attributes that give value to a commodity. Flavor, texture, nutrition, and shelf life are key quality traits that ensure market value and consumer acceptance. In pear fruit, soluble sugars, organic acids, amino acids, and total flavonoids contribute to flavor and overall quality. Transcription factors (TFs) regulate the accumulation of these metabolites during development or in response to the environment. Here, we report a novel TF, PpbZIP44, as a positive regulator of primary and secondary metabolism in pear fruit. Analysis of the transient overexpression or RNAi-transformed pear fruits and stable transgenic tomato fruits under the control of the fruit-specific E8 promoter demonstrated that PpZIP44 substantially affected the contents of soluble sugar, organic acids, amino acids, and flavonoids. In E8::PpbZIP44 tomato fruit, genes involved in carbohydrate metabolism, amino acid, and flavonoids biosynthesis were significantly induced. Furthermore, in PpbZIP44 overexpression or antisense pear fruits, the expression of genes in the related pathways was significantly impacted. PpbZIP44 directly interacted with the promoter of PpSDH9 and PpProDH1 to induce their expression, thereby depleting sorbitol and proline, decreasing citrate and malate, and enhancing fructose contents. PpbZIP44 also directly bound to the PpADT and PpF3H promoters, which led to the carbon flux toward phenylalanine metabolites and enhanced phenylalanine and flavonoid contents. These findings demonstrate that PpbZIP44 mediates multimetabolism reprogramming by regulating the gene expression related to fruit quality compounds.

The DR5 and E8 reporters are suitable systems for studying the application of the <i>Ruby</i> reporter gene in tomato

<sec><title/> Tomato (<italic>Solanum lycopersicum</italic>) fruits can be found in a range of colors, including green, yellow, red, pink and purple. The various colors are determined by the accumulation of molecules such as chlorophyll, carotenoids (including lycopene) and flavonoids. Since the DR5 promoter is induced by auxin, we can infer that the auxin responses differ at different developmental stages in tomato. Fruit-specific promoters can effectively drive gene expression in fruits, and the E8 promoter is one of the most widely used fruit-specific promoters. In this study, DR5::<italic>Ruby</italic> transgenic tomato plants were used to further clarify the auxin content at different developmental stages in tomato. The results showed that the nonuniformity of <italic>Ruby</italic> activity was consistent with the natural distribution characteristics of native auxin, and the results were also consistent with the qRT‒PCR results. Furthermore, the accumulation of endogenous auxin at the immature green stage (IMG) was higher than that at the other stages. </sec><sec><title/> In addition, E8::<italic>Ruby</italic> was transformed into different tomato varieties (red, white and yellow) to generate new tomato varieties with fruit of different colors. The results showed that the E8::<italic>Ruby-</italic>transformed plants exhibited asymmetrical pink coloration in different tomato tissues of plants from different tomato cultivars. In conclusion, all the results showed that DR5 and E8 are suitable systems for studying the application of the <italic>Ruby</italic> reporter gene in tomato. Moreover, the study will provide insight for the improvement of tomato fruit color via biotechnology. </sec>

Expression of B. subtilis Phytase gene driven by fruit specific E8 promoter for enhanced minerals, metabolites and phytonutrient in cucumber fruit

The fruit nutrigenomics is an interesting and important research area towards nutrition enhancement. The phytic acid is one of the major antinutrient compound, present in seeded fruits and crops. It hinders the absorption of iron (Fe), zinc (Zn), magnesium (Mg), potassium (K) and calcium (Ca), causing mineral deficiencies. In the present study, the BsPhy gene was overexpressed in the cucumber fruits using the tomato fruit specific E8 and constitutive CaMV 35S promoter. The E8 promoter imparted heterologous expression of GUS gene in cucumber fruits, furthermore, the fruit specific expression of E8 promoter with BsPhy gene was confirmed in transgenics (E8::BsPhy) using anti rabbit-phytase antibody. The physio-biochemical analysis of transgenics revealed, maximum phytase activity in E8::BsPhy cucumber fruits at 10 days after anthesis (DAA) compared to 35S::BsPhy and wild-type (WT) fruits. Consequently, E8::BsPhy fruits also showed increased amount of inorganic phosphorus (Pi), total phosphorus (P), minerals (Zn, Fe, Mg, K, Ca), total carotenoid and other macronutrients at 10 DAA compared to 35S::BsPhy fruits. The metabolite profiling of fruits (10 DAA) showed increased sugars, amino acids, sugar acids and polyols, in both E8::BsPhy and 35S::BsPhy transgenics suggesting higher phytate metabolism, compared to WT fruits. Interestingly, both the transgenic fruits showed higher fruit biomass and yield along with improved nutritional quality, which can be attributed to increased P and Zn contents in transgenic fruits, compared to WT fruits. Our findings reveal that the BsPhy gene enhances minerals and macronutrients in transgenic cucumber fruits making it nutritious and healthy.

In silico Analysis of Polyamine Rich Transgenic Tomato Fruit Transcriptome for Salicylic Acid Biosynthesis

EFFECT of higher levels of polyamines (PAs), spermidine (SPD) and spermine (SPM), were evaluated on the salicylic acid biosynthesis genes. It has been previously reported that higher steady state levels of a pathogenesis protein pR1b1, a salicylic acid (SA) regulated protein, are present in tomato fruits with higher levels of anabolic biogenic amines, spermidine (SPD) and spermine (SPM) resulting from the expression of yeast S-adenosylmethionine decarboxylase (SAMDC), under a fruit specific E8 promoter in ripening tomato fruit. Based on this observation, we hypothesized that high SPD/SPM fruit would enhance the expression of SA- biosynthesis genes leading to enhanced SA production. In plants, SA is synthesized either through phenylalanine ammonia-lyase (PAL) or isochorismate synthase (ICS) pathway. We identified ten putative genes for the SA biosynthesis pathways in tomato and determined the relative abundance of their transcript based on RNAseq transcriptomic analyses of mature green and turning stage tomato fruits from WT and two isogenic independent SAMDC-transgenic homozygous lines with higher levels of SPD/SPM in ripening fruits. We show that at MG stage the transgenic fruits exhibited higher steady state levels of transcripts of CS, PAL1-7, ICS, IPL/PRXR1, CS, and PAL 4-10, but lower levels for CM1, CM2, EPS1, and PBS3-2. At the turning stage of tomato fruit ripening the steady state levels of only CS and PBS3-2 were upregulated whereas the transcript levels of CM1, CM2, PaL1-4AAO, PBS3-1and EPS1 were down regulated. Taken together these results suggest that SPD/SPM play role in the SA biosynthesis and higher levels of various genes especially in in PAL pathway likely increased the SA levels in the transgenic fruits.

A Transcriptional Network Makes Normal Tomato Fruit Not Purple

A Transcriptional Network Makes Normal Tomato Fruit Not Purple

Stochastic Modeling of the Co-Regulation between Early and E8 Promoters in Human Papillomavirus.

High risk HPV can induce cervical and oropharyngeal cancerous Iesions. The initial phase of the infection is characterized by a fine regulation of the viral DNA replication, in order to maintain 10-100 DNA copies per cell. Such regulation is primarily controlled by El and E2 proteins produced by the early promoter. The recently discovered E8 promoter is capable to co-regulate the early one in order to maintain a low and constant viral DNA copy number.The aim of this study is to develop a novel stochastic mathematical model of the co-regulation between the E8 and the early promoter, with the main purpose to rigorously show the E8 promoter capability to finely regulate the HPV transcripts which control the DNA replication in the first stages of the infection.The model, condensing the biological knowledge present in literature, describes the interaction between the two promoters and shows how the E8 co-regulation is capable to reject the stochastic noise of E2 gene expression to a higher extent than the early promoter negative auto-feedback. This proves the capability of the E8 promoter to finely control the HPV genomes copy number.

Detection and Amplification of the E8 Promoter in Tomato Plant and Sequencing of the E8 Gene

Tomato (Lycopersicon esculentum Mill) is edible, often red berry-type fruit and a rich source of Fe, Vitamin A, B and C. It is widely cultivated all over the world. E8 gene is related to ethylene biosynthesis in plants. Ethylene is responsible for growth and ripening of fruits. The present study was carried out to detect and amplify the E8 Promoter in the leaves and fruits of Tomato, Spring onion, Capsicum and Lettuce. The CTAB Protocol of Doyle and Doyle (1990) were used for DNA extraction from leaves and fruits and quality was checked on gel electrophoresis. PCR program for E8 was optimized and PCR products were sequenced. The sequencing results were aligned and Blast. The result indicated that in Tomato fruit the E8 gene is detected and amplified as compare to leaves and other plants. This E8 primer will be utilized for different fruits as well.

Detection and Amplification of the E8 Promoter in Tomato Plant and Sequencing of the E8 Gene

Tomato (Lycopersicon esculentum Mill) is edible, often red berry-type fruit and a rich source of Fe, Vitamin A, B and C. It is widely cultivated all over the world. E8 gene is related to ethylene biosynthesis in plants. Ethylene is responsible for growth and ripening of fruits. The present study was carried out to detect and amplify the E8 Promoter in the leaves and fruits of Tomato, Spring onion, Capsicum and Lettuce. The CTAB Protocol of Doyle and Doyle (1990) were used for DNA extraction from leaves and fruits and quality was checked on gel electrophoresis. PCR program for E8 was optimized and PCR products were sequenced. The sequencing results were aligned and Blast. The result indicated that in Tomato fruit the E8 gene is detected and amplified as compare to leaves and other plants. This E8 primer will be utilized for different fruits as well.

Activating glutamate decarboxylase activity by removing the autoinhibitory domain leads to hyper γ-aminobutyric acid (GABA) accumulation in tomato fruit.

The C-terminal extension region of SlGAD3 is likely involved in autoinhibition, and removing this domain increases GABA levels in tomato fruits. γ-Aminobutyric acid (GABA) is a ubiquitous non-protein amino acid with several health-promoting benefits. In many plants including tomato, GABA is synthesized via decarboxylation of glutamate in a reaction catalyzed by glutamate decarboxylase (GAD), which generally contains a C-terminal autoinhibitory domain. We previously generated transgenic tomato plants in which tomato GAD3 (SlGAD3) was expressed using the 35S promoter/NOS terminator expression cassette (35S-SlGAD3-NOS), yielding a four- to fivefold increase in GABA levels in red-ripe fruits compared to the control. In this study, to further increase GABA accumulation in tomato fruits, we expressed SlGAD3 with (SlGAD3 OX ) or without (SlGAD3ΔC OX ) a putative autoinhibitory domain in tomato using the fruit ripening-specificE8 promoter and the Arabidopsis heat shock protein 18.2 (HSP) terminator. Although the GABA levels in SlGAD3 OX fruits were equivalent to those in 35S-SlGAD3-NOS fruits, GABA levels in SlGAD3ΔC OX fruits increased by 11- to 18-fold compared to control plants, indicating that removing the autoinhibitory domain increases GABA biosynthesis activity. Furthermore, the increased GABA levels were accompanied by a drastic reduction in glutamate and aspartate levels, indicating that enhanced GABA biosynthesis affects amino acid metabolism in ripe-fruits. Moreover, SlGAD3ΔC OX fruits exhibited an orange-ripe phenotype, which was associated with reduced levels of both carotenoid and mRNA transcripts of ethylene-responsive carotenogenic genes, suggesting that over activation of GAD influences ethylene sensitivity. Our strategy utilizing the E8 promoter and HSP terminator expression cassette, together with SlGAD3 C-terminal deletion, would facilitate the production of tomato fruits with increased GABA levels.

Phosphoenolpyruvate carboxykinase (PEPCK) deficiency affects the germination, growth and fruit sugar content in tomato (Solanum lycopersicum L.)

Phosphoenolpyruvate carboxykinase (PEPCK) is a key regulatory enzyme and is utilized in the gluconeogenesis pathway in plants. Although, its catalytic and regulatory properties are quite well understood, there are uncertainties regarding its physiological role in many plants tissues such as the flesh of developing fruits. To further understand the function of PEPCK in fruits and other tissues, RNAi transgenic tomato plants in which SlPEPCK transcription was down-regulated by either CaMV 35S constitutive promoter or the fruit-specific E8 promoter were generated and characterized on the basis of their phenotypic and metabolic aspects. In the PEPCK-deficient lines, prominent growth suppression of germinated seedlings was observed and other vegetative suppression appeared during the early stage of plant growth in the 35S promoter-driven lines. In particular, root elongation was most obviously suppressed in the germinated seedlings, indicating that the gluconeogenesis pathway is involved in the root growth of seedlings. Regarding the primary metabolism in fruit, the soluble sugar content tended to decrease, whereas the malate content tended to increase in ripening fruits of the RNAi lines compared with the wild type. These results indicate that activation of the gluconeogenesis pathway from organic acids to sugars occurs during ripening but is suppressed by the knocking down of the PEPCK gene, suggesting that PEPCK participates in determining the sugar/acid ratio in ripening fruit.

Characterization of the Human Papillomavirus 16 E8 Promoter.

Persistent infections with certain human papillomaviruses (HPV) such as HPV16 are a necessary risk factor for the development of anogenital and oropharyngeal cancers. HPV16 genomes replicate as low-copy-number plasmids in the nucleus of undifferentiated keratinocytes, which requires the viral E1 and E2 replication proteins. The HPV16 E8^E2C (or E8^E2) protein limits genome replication by repressing both viral transcription and the E1/E2-dependent DNA replication. How E8^E2C expression is regulated is not understood. Previous transcript analyses indicated that the spliced E8^E2C RNA is initiated at a promoter located in the E1 region upstream of the E8 gene. Deletion and mutational analyses of the E8 promoter region identify two conserved elements that are required for basal promoter activity in HPV-negative keratinocytes. In contrast, the transcriptional enhancer in the upstream regulatory region of HPV16 does not modulate basal E8 promoter activity. Cotransfection studies indicate that E8^E2C inhibits, whereas E2 weakly activates, the E8 promoter. Interestingly, the cotransfection of E1 and E2 induces the E8 promoter much more strongly than the major early promoter, and this is partially dependent upon binding of E2 to Brd4. Mutation of E8 promoter elements in the context of HPV16 genomes results in an increased genome copy number and elevated levels of viral early and late transcripts. In summary, the promoter responsible for the expression of E8^E2C is both positively and negatively regulated by viral and cellular factors, and this regulatory circuit may be crucial to maintain a low but constant copy number of HPV16 genomes in undifferentiated cells. HPV16 replicates in differentiating epithelia and can cause cancer. How HPV16 maintains its genome in undifferentiated cells at a low but constant level is not well understood but may be relevant for the immunological escape of HPV16 in the basal layers of the infected epithelium. This study demonstrates that the expression of the viral E8^E2C protein, which is a potent inhibitor of viral replication in undifferentiated cells, is driven by a separate promoter. The E8 promoter is both positively and negatively regulated by viral proteins and thus most likely acts as a sensor and modulator of viral copy number.

Transient expression of optimized and synthesized nattokinase gene in melon (&lt;i&gt;Cucumis melo&lt;/i&gt; L.) fruit by agroinfiltration

A synthetic gene (sNK) encoding Nattokinase (NK) was constructed by modifying its sequence based on the codon usage in plants. A version (sNKi) incorporating the first intron from the tomato E8 gene was also constructed. The synthetic sNK and sNKi genes were transiently expressed in melon (Cucumis melo L.) fruit. Quantitative real-time reverse transcription PCR (qRT-PCR) analysis and fibrinolytic activity assays showed that the expression level of recombinant NK controlled by E8 promoter was higher than that controlled by 35S promoter, with the maximum fibrinolytic activity of 79.30 U ml−1. The intron could enhance the expression of sNK to an extent of 27.42%. Orthogonal tests determined the optimal agroinfiltration as: an acetosyringone concentration of 0.25 mM, an OD600=0.6 and harvesting 5 days after infiltration.

Cloning and analysing of cis-acting elements in the E8 promoter from tomato (Lycopersicon esculentum L.)

Promoter is a sequence located at the 5’ end upstream of the gene, that plays a key role in gene expression. In tomato, the E8 gene is highly expressed during fruit ripening, and is controlled by E8 promoter. E8 promoter includes the cis-acting elements responding to ethylene or receiving independent signals of ethylene for fruit ripening. This study presents results of E8 promoter cloning and sequencing from the tomato cultivar PT18 for construction of vector to express target gene in tomato. The obtained data showed that the E8 promoter has 2203 bp in length and carries a full range of cis-acting elements similar to a typical promoter such as TATA box, box CAAT and GATA box. Furthermore, this promoter contains two fruit-ripening regulatory regions. Nucleotide sequences of E8 promoter had high similarity when compared with other E8 promoter sequences in Genebank. In addition, the nucleotide sequence of E8 promoter was submitted in Genebank with the accession number KJ561284. The results is the foundation that will contribute a fundamental basis for further researches in order to construct expression vector carrying E8 promoter in gene transfer of plants.

Cloning and analysing of cis-acting elements in the E8 promoter from tomato (Lycopersicon esculentum L.)

Promoter is a sequence located at the 5’ end upstream of the gene, that plays a key role in gene expression. In tomato, the E8 gene is highly expressed during fruit ripening, and is controlled by E8 promoter. E8 promoter includes the cis-acting elements responding to ethylene or receiving independent signals of ethylene for fruit ripening. This study presents results of E8 promoter cloning and sequencing from the tomato cultivar PT18 for construction of vector to express target gene in tomato. The obtained data showed that the E8 promoter has 2203 bp in length and carries a full range of cis-acting elements similar to a typical promoter such as TATA box, box CAAT and GATA box. Furthermore, this promoter contains two fruit-ripening regulatory regions. Nucleotide sequences of E8 promoter had high similarity when compared with other E8 promoter sequences in Genebank. In addition, the nucleotide sequence of E8 promoter was submitted in Genebank with the accession number KJ561284. The results is the foundation that will contribute a fundamental basis for further researches in order to construct expression vector carrying E8 promoter in gene transfer of plants.

Isolation and cloning of fruit-specific E8 promoter from local small fruit tomato (Lycopersicon esculentum L.) and analysis its transient expression

Isolation and characterization of specific promoter is important for efficient expression of recombinant proteins in plants. E8 is a fruit specific promoter with 2.2 kb length. To isolate E8 fruit specific promoter, seeds of local small fruit Sardasht genotype were sown in greenhouse and genomic DNA was extracted from young leaves using CTAB method. The promoter region was amplified using specific primers designed based on conserved regions of the sequences available at NCBI database. The amplified fragments ligated into PTZ57R/T vector and transferred into competence cells of DH5α strain of E. coli for cloning. Plasmid was extracted after confirming the presence of the inserted segments in plasmid based on colony PCR test and enzyme digestion. The extracted plasmids were used for sequencing the fragments. Sequence analysis revealed successful isolation of E8 promoter segment. To analyze the activity promoter in tomato tissues, the CaMV35S promoter in pBI121 binary vector replaced by E8 promoter and transferred to GV3101 strain of Agrobacterium. Transient expression was assessed using gus gene under E8 promoter with aim of transgenic Agrobacterium agro-infiltrated into fruit tissue. Presence of indigo color indicated successful expression of gus reporter gene under E8 promoter at the cells receipt the gene construct.

Construction of Expression Vector with Fruit-Specific Promoter and Genetic Transformation of Strawberry

In order to transcription and expression an exogenous gene just in transgenic strawberry fruit, the gene encoding tomato fruit-specific E8 promoter was cloned. We constructed plant expression vector with E8 promoter replace the CaMV35S of pBI121 that used for transformation of strawberry. Recombination plasmid was identified by PCR, restriction enzymes digestion and sequencing analysis, and transfered into the agrobacterium EHA105 strain. At the same time, the agrobacterium EHA105, transformation and no transformation tissue of strawberry used as the experiment materials to study the expression of reporter genegusAby the histochemical staining method. The results show that agrobacterium which containsgusAgene and strawberry organization of transformation can be dyed blue, others were not stain. So in testing the transgenic plants by agroinfiltration transient expression system can reduce or avoid false positive appearance.

An E8 promoter–HSP terminator cassette promotes the high-level accumulation of recombinant protein predominantly in transgenic tomato fruits: a case study of miraculin

The E8 promoter-HSP terminator expression cassette is a powerful tool for increasing the accumulation of recombinant protein in a ripening tomato fruit. Strong, tissue-specific transgene expression is a desirable feature in transgenic plants to allow the production of variable recombinant proteins. The expression vector is a key tool to control the expression level and site of transgene and recombinant protein expression in transgenic plants. The combination of the E8 promoter, a fruit-ripening specific promoter, and a heat shock protein (HSP) terminator, derived from heat shock protein 18.2 of Arabidopsis thaliana, produces the strong and fruit-specific accumulation of recombinant miraculin in transgenic tomato. Miraculin gene expression was driven by an E8 promoter and HSP terminator cassette (E8-MIR-HSP) in transgenic tomato plants, and the miraculin concentration was the highest in the ripening fruits, representing 30-630μg miraculin of the gram fresh weight. The highest level of miraculin concentration among the transgenic tomato plant lines containing the E8-MIR-HSP cassette was approximately four times higher than those observed in a previous study using a constitutive 35S promoter and NOS terminator cassette (Hiwasa-Tanase et al. in Plant Cell Rep 30:113-124, 2011). These results demonstrate that the combination of the E8 promoter and HSP terminator cassette is a useful tool to increase markedly the accumulation of recombinant proteins in a ripening fruit-specific manner.

Alteration of the Interconversion of Pyruvate and Malate in the Plastid or Cytosol of Ripening Tomato Fruit Invokes Diverse Consequences on Sugar But Similar Effects on Cellular Organic Acid, Metabolism, and Transitory Starch Accumulation

The aim of this work was to investigate the effect of decreased cytosolic phosphoenolpyruvate carboxykinase (PEPCK) and plastidic NADP-dependent malic enzyme (ME) on tomato (Solanum lycopersicum) ripening. Transgenic tomato plants with strongly reduced levels of PEPCK and plastidic NADP-ME were generated by RNA interference gene silencing under the control of a ripening-specific E8 promoter. While these genetic modifications had relatively little effect on the total fruit yield and size, they had strong effects on fruit metabolism. Both transformants were characterized by lower levels of starch at breaker stage. Analysis of the activation state of ADP-glucose pyrophosphorylase correlated with the decrease of starch in both transformants, which suggests that it is due to an altered cellular redox status. Moreover, metabolic profiling and feeding experiments involving positionally labeled glucoses of fruits lacking in plastidic NADP-ME and cytosolic PEPCK activities revealed differential changes in overall respiration rates and tricarboxylic acid (TCA) cycle flux. Inactivation of cytosolic PEPCK affected the respiration rate, which suggests that an excess of oxaloacetate is converted to aspartate and reintroduced in the TCA cycle via 2-oxoglutarate/glutamate. On the other hand, the plastidic NADP-ME antisense lines were characterized by no changes in respiration rates and TCA cycle flux, which together with increases of pyruvate kinase and phosphoenolpyruvate carboxylase activities indicate that pyruvate is supplied through these enzymes to the TCA cycle. These results are discussed in the context of current models of the importance of malate during tomato fruit ripening.

Fruit‐specific overexpression of wound‐induced tap1 under E8 promoter in tomato confers resistance to fungal pathogens at ripening stage

Based on high economic importance and nutritious value of tomato fruits and as previous studies employed E8 promoter in fruit ripening-specific gene expression, we have developed transgenic tomato plants overexpressing tomato anionic peroxidase cDNA (tap1) under E8 promoter. Stable transgene integration was confirmed by polymerase chain reaction (PCR) and Southern analysis for nptII. Northern blotting confirmed elevated tap1 levels in the breaker- and red-ripe stages of T(1) transgenic fruits, whereas wild-type (WT) plants did not show tap1 expression in these developmental stages. Further, tap1 expression levels were significantly enhanced in response to wounding in breaker- and red-ripe stages of transgenic fruits, whereas wound-induced expression of tap1 was not detected in WT fruits. Confocal microscopy revealed high accumulation of phenolic compounds at the wound site in transgenic fruits suggesting a role of tap1 in wound-induced phenolic polymerization. Total peroxidase activity has increased remarkably in transgenic pericarp tissues in response to wounding, while very less or minimal levels were recorded in WT pericarp tissues. Transgenic fruits also displayed reduced post-harvest decay and increased resistance toward Alternaria alternata and Fusarium solani infection with noticeable inhibition in lesion formation. Conidiospore germination and mycelial growth of F. solani were severely inhibited when treated with E8-tap1 fruit extracts compared to WT fruits. 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay showed reduced spore viability when incubated in E8-tap1 fruit extracts. Thus, fruit-specific expression of tap1 using E8 promoter is associated with enhanced total peroxidase activity and high phenolic accumulation in fruits with minimized post-harvest deterioration caused by wounding and fungal attack in tomato fruits.

Novel promoters that induce specific transgene expression during the green to ripening stages of tomato fruit development

Fruit-specific promoters have been used as genetic engineering tools for studies on molecular mechanism of fruit development and advance in fruit quality and additional value by increasing functional component. Especially fruit-ripening specific promoters have been well utilized and studied in tomato; however, few studies have reported the development of promoters that act at fruit developing stages such as immature green and mature green periods. In this study, we report novel promoters for gene expression during the green to ripening stages of tomato fruit development. Genes specifically expressed at tomato fruit were selected using microarray data. Subsequent to confirmation of the expression of the selected 12 genes, upstream DNA fragments of the genes LA22CD07, Les.3122.2.A1_a_at and LesAffx.6852.1.S1_at which specifically expressed at fruit were isolated from tomato genomic DNA as promoter regions. Isolated promoter regions were fused with the GUS gene and the resultant constructs were introduced into tomato by agrobacterium-mediated transformation for evaluation of promoter activity in tomato fruit. The two promoters of LA22CD07, and LesAffx.6852.1.S1_at showed strong activity in the fruit, weak activity in the flower and undetectable activity in other tissues. Unlike well-known fruit-ripening specific promoters, such as the E8 promoter, these promoters exhibited strong activity in green fruit in addition to red-ripening fruit, indicating that the promoters are suitable for transgene expression during green to ripening stages of tomato fruit development. Novel fruit-specific promoters have been identified and are suitable for transgene expression during green to ripening stages of tomato fruit development.